title: Antiparasitic effect of a fraction enriched in tight-binding protease inhibitors isolated from the Caribbean coral Plexaura homomalla.
abstract: Malaria and American Trypanosomiasis constitute major global health problems. The continued emergence and spreading of resistant strains and the limited efficacy and/or safety of currently available therapeutic agents require a constant search for new sources of antiparasitic compounds. In the present study, a fraction enriched in tight-binding protease inhibitors was isolated from the Caribbean coral Plexaura homomalla (Esper, 1792), functionally characterized and tested for their antiparasitic activity against Trypanosoma cruzi and Plasmodium falciparum. The resultant fraction was chromatographically enriched in tight-binding inhibitors active against Papain-like cysteine peptidases (92%) and Pepsin-like aspartyl peptidases (8%). Globally, the inhibitors present in the enriched fraction showed no competition with substrates and apparent Ki values of 1.99 and 4.81nM for Falcipain 2 and Cruzipain, the major cysteine peptidases from P. falciparum and T. cruzi, respectively. The inhibitor-enriched fraction showed promising antiparasitic activity in cultures. It reduced the growth of the chloroquine-resistant P. falciparum strain Dd2 (IC50=0.46μM) and promoted the apparent accumulation of trophozoites, both consistent with a blockade in the hemoglobin degradation pathway. At sub-micromolar concentrations, the inhibitor-enriched fraction reduced the infection of VERO cells by T. cruzi (CL Brener clone) trypomastigotes and interfered with intracellular differentiation and/or replication of the parasites. This study provides new scientific evidence that confirms P. homomalla as an excellent source of tight-biding protease inhibitors for different proteases with biomedical relevance, and suggests that either the individual inhibitors or the enriched fraction itself could be valuable as antiparasitic compounds.
authors.affiliation: Centro de Estudio de Proteínas, Facultad de Biología, Universidad de la Habana, 25 #455 Entre J e I, La Habana, Cuba; Red CYTED-PROMAL (210RT0398), Proteómica y Quimiogenómica de Inhibidores de Proteasas de Origen Natural con Potencial Terapéutico en Malaria, Universidad Nacional de la Plata, La Plata, Argentina. Electronic address: emirsalas@gmail.com.
authors.forename: Emir
authors.initials: E
authors.lastname: Salas-Sarduy
authors.forename: Aymara
authors.initials: A
authors.lastname: Cabrera-Muñoz
authors.forename: Ana
authors.initials: A
authors.lastname: Cauerhff
authors.forename: Yamile
authors.initials: Y
authors.lastname: González-González
authors.forename: Sebastián A
authors.initials: SA
authors.lastname: Trejo
authors.forename: Agustina
authors.initials: A
authors.lastname: Chidichimo
authors.forename: Maria de Los Angeles
authors.initials: Mde L
authors.lastname: Chávez-Planes
authors.forename: Juan José
authors.initials: JJ
authors.lastname: Cazzulo
mesh_terms: D000818:Animals; D037421:Anthozoa / Q000737:chemistry*; D000981:Antiprotozoal Agents / Q000302:isolation & purification / Q000494:pharmacology*; D002417:Cattle; D002522:Chlorocebus aethiops; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000469:parasitology; D006801:Humans; D020128:Inhibitory Concentration 50; D010206:Papain / Q000037:antagonists & inhibitors / Q000378:metabolism; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000201:enzymology / Q000254:growth & development; D015800:Protozoan Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine; D014349:Trypanosoma cruzi / Q000187:drug effects* / Q000201:enzymology / Q000254:growth & development; D014709:Vero Cells
chemical_list: D000981:Antiprotozoal Agents; D015853:Cysteine Proteinase Inhibitors; D015800:Protozoan Proteins; D003546:Cysteine Endopeptidases; C416224:falcipain 2; D010206:Papain; C063214:cruzipain
keywords: 7-amino-4-methyl coumarin; AAFP; ACN; AMC; AU; Antiparasitic agents; BAPA; CMD; Cysteine peptidases; E-64; IF; N-(4-metoxiphenylazoformyl)-L-phenylalanine; NaAc; Plasmodium falciparum; Plexaura homomalla; RP; RT; TFA; Tight-binding inhibitor; Trypanosoma cruzi; Z-FR-AMC; absorbance units; acetonitrile; benzoyl-arginyl-p-nitro-anilide-HCl; benzyloxycarbonyl-Phenyl-Arginyl-7-amino-4-methyl coumarin; carboxymethyl dextran; intensity fading; reverse-phase; room temperature; sodium acetate buffer; trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane; trifluoracetic acid